Abstract Abstract Individuals who use injection drugs are among those at highest risk for HIV-1 infection, and their relative contribution to the total number of HIV-1-infected persons is increasing worldwide. This is particularly true for the roughly 2 million individuals affected by the opioid crisis in the US, of who approximately 9% (180,000 persons) are currently estimated to be HIV-1-infected. Owing to recent advances in improving access to care and adherence to treatment, a considerable proportion of these individuals is now able to maintain undetectable viral loads during ongoing use of opioids (oxycodone, heroin, hydromorphone, fentanyl) or opioid substitution agents (methadone or buprenorphine). However, residual reservoirs of virally infected cells persist in these patients, and represent the main barrier against a long-lasting drug-free remission of viral infection. Recently, there is substantial progress in understanding the cellular compartments and mechanisms of viral reservoir persistence, but opioid addicts were either highly underrepresented or entirely excluded from such investigations. Yet, there are reasons to believe that the size, structure and composition of the viral reservoir in opioid users is substantially different from HIV-1-infected individuals who do not use drugs. For instance, opioid drug abuse can profoundly change gene expression patterns and also induces epigenetic chromatin modifications, both of which are known to affect the susceptibility to retroviral infection, the selection of chromosomal integration sites and the transcriptional activity of integrated HIV-1 proviruses. This project sets out to conduct a detailed analysis of the viral reservoir structure and composition in HIV-1-infected opioid drug addicts, using a spectrum of novel next- generation sequencing technologies allowing to profile the viral reservoir at a previously unprecedented breadth and depth. In Specific Aim 1, we will comprehensively analyze the chromosomal location of intact HIV-1 proviruses, based on a novel experimental approach combining full-genome amplification, near full-length viral sequencing and ligation-mediated PCR for chromosomal integration site analysis. Subsequently, we will use ATAC-Seq and RNA-Seq to characterize the chromatin accessibility and transcriptional activity of genes harboring intact proviruses (Specific Aim 2), determine innate and adaptive immune responses correlated with the intact proviral reservoirs (Specific Aim 3), and investigate epigenetic features associated with intact proviral reservoir persistence (Specific Aim 4) in HIV-1-infected opioid addicts and a control cohort of HIV-1 patients without past or present drug abuse. Together, these studies will provide a wealth of information for developing targeted interventions to reduce HIV-1 persistence during antiretroviral therapy in HIV-1-infected individuals who use opioids.